Here we report our novel observation that parkin interacts with and is phosphorylated at tyrosine-143 by c-Abl. Activation of c-Abl and parkin tyrosine phosphorylation occur after oxidative and dopamine stress both in vitro and in vivo, causing significant loss of parkin's ubiquitin E3-ligase activity and leading to accumulation of neurotoxic AIMP2 and FBP-1, ultimately compromising parkin's protective function (see scheme, ). STI-571, a selective c-Abl inhibitor, prevented parkin tyrosine phosphorylation, preserved its E3-ligase activity and cytoprotective function. The protective effect of STI-571 was parkin-dependent, since shRNA knockdown of parkin specifically attenuated STI-571 protection. Moreover, we observed tyrosine phosphorylation of c-Abl and parkin, along with accumulation of toxic parkin substrates, AIMP2 (p38/JTV-1) and FBP-1, in nigrostriatum of PD patients. There was significant correlation among tyrosine-phosphorylated parkin, activated c-Abl, and AIMP2 and FBP-1 levels in striatum of PD patients. These data provide convincing evidence for a novel oxidative stress-induced cell signaling pathway that negatively regulates parkin function through c-Abl-mediated tyrosine phosphorylation and may contribute to nigrostriatal neuronal injury and disease progression in sporadic PD.
Recently, it has been reported that oxidative (Wang et al., 2005
), nitrosative (Chung et al., 2004
; Yao et al., 2004
), and dopaminergic (LaVoie et al., 2005
) stress impair parkin function by direct modification and/or through alteration in parkin solubility, thus linking parkin to sporadic PD. However, the mechanisms underlying parkin inactivation have remained unclear. Our data provide a molecular mechanism for parkin inactivation, and support a role of parkin in pathogenesis of more common sporadic form of PD. Thus, oxidative and dopamine-stress lead to c-Abl activation, parkin tyrosine phosphorylation and the consequent loss of parkin ubiquitination-dependent cytoprotective function. c-Abl-mediated parkin inactivation in response to oxidative and dopaminergic stress seems to be the dominant pathway induced by these stressors, since the c-Abl inhibitor, STI-571, blocked inactivation of parkin.
Attempts to characterize tyrosine phosphorylation of parkin by capillary HPLC electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) both in vitro and in vivo were unsuccessful, despite the ability to detect the non-phosphorylated peptide in both the precursor and targeted product scans (data not shown). We suspect that detection of Y143 phospho-peptide via MS/MS is not technically feasible due to poor solubility, since parkin peptides containing phosphorylated Y143 failed to dissolve in solvents utilized in the MS/MS analysis (data not shown). Since we were unable to prove definitively via mass-spectrometry that parkin is tyrosine-phosphorylated at Y143, we cannot exclude the possibility that there are additional c-Abl targets that may contribute to the pathogenesis of PD.
Our finding that this pathway is seen predominantly in the striatum suggests that dopamine-containing cells of the nigrostriatum are particularly predisposed. c-Abl activation and parkin tyrosine phosphorylation appear to reflect processes that are unique to nigrostriatum and not necessarily associated with inclusion bodies, since we did not observe c-Abl activation and tyrosine phosphorylation of parkin in the cortex, even in the four PD patients with neocortical Lewy bodies. Furthermore, parkin tyrosine phosphorylation and AIMP2 accumulation in striatum compared with cortex appears to be associated with increased oxidative stress in the striatum of PD patients, as indicated by OxyBlot analysis. Since oxidative stress is intimately involved in sporadic PD, we propose a novel stress-induced cell signaling mechanism featuring activated c-Abl, which inhibits parkin function and consequently increases cell death due to accumulation of cytotoxic parkin substrates, such as AIMP2 ().
The c-Abl inhibitor STI-571 is widely used chemotherapeutic agent for chronic myelogenous leukemia. The finding that STI-571 inhibits c-Abl's deleterious effects on parkin by preventing it's phosphorylation and preserving its protective function, holds promise for further testing of this agent as a neuroprotective therapeutic for PD. Since STI-571 has limited brain bioavailability (Breedveld et al., 2005
), the amount of protection afforded by inhibition of c-Abl in vivo
may be greatly improved by using related compounds with enhanced brain penetration. The identification of c-Abl tyrosine phosphorylation-mediated inhibition of parkin activity and its pathological relevance as demonstrated in PD will pave the way for better understanding of the pathophysiology of this disease.